Genetic engineering of plant chloroplasts

ABSTRACT

Novel chimeric constructions and methods for their use are provided for expression of exogenous genes in a plant chloroplast. Particularly, expression is achieved by the use of a chloroplast or bacterial 5′ untranslated region in the expression cassette. The expression cassette may be integrated into the chloroplast genome by the use of chloroplast DNA flanking sequences, or may replicate autonomously if provided with a chloroplast origin of replication. Plants and cells containing the transformed chloroplasts are also provided. The constructs may be used with both monocotyledenous and dicotyledenous chloroplasts.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. patentapplication Ser. No. 249,616 filed Sep. 26, 1988 which disclosure ishereby incorporated by reference.

INTRODUCTION TECHNICAL FIELD

[0002] This invention relates to methods and compositions fortransformation of plant chloroplasts as well as the resulting cells andplants containing transformed chloroplasts.

BACKGROUND

[0003] Many techniques have been proposed for the transfer of DNA toplants such as direct DNA uptake, microinjection of pure DNA and the useof viral or plasmid vectors. The strategies for gene transfer involvethe introduction of foreign DNA into cells or protoplasts followed byintegration into the nuclear genome. However, eukaryotic cells, moreparticularly plant cells, contain distinct subcellular compartments ororganelles delimited by characteristic membrane systems which performspecialized functions within the cell.

[0004] In photosynthetic leaf cells of higher plants the mostconspicuous organelles are the chloroplasts, which exist in asemi-autonomous fashion within the cell, containing their own geneticsystem and protein synthesis machinery, but relying upon a closecooperation with the nucleo-cytoplasmic system in their development andbiosynthetic activities. The chloroplast present in leaf cells is onedevelopment stage of this organelle. Proplastids, etioplasts,amyloplasts, and chromoplasts are different stages. The embodiments ofthis invention apply to the organelle “at large” which will be referredto as a “chloroplast”.

[0005] The most essential function of chloroplasts is the performance ofthe light-driven reactions of photosynthesis including fixation ofcarbon dioxide. However, chloroplasts carry out many other biosyntheticprocesses of importance to the plant cell, such as synthesis of fattyacids. In addition, the reducing power of light-activated electronsdrives the reduction of nitrites (NO₂ ⁻) to ammonia (NH₃) in thechloroplast; this ammonia provides the plant with nitrogen required forthe synthesis of amino acids compartmentalized in the chloroplast andnucleotides.

[0006] Other functions in which the chloroplast is involved are ofinterest to the agriculture industry. For example, many herbicides actby blocking functions which are performed within the chloroplast.Triazine derived herbicides inhibit photosynthesis by displacing aplastoquinone molecule from its binding site in the 32 kDa polypeptideof photo system II. This 32 kDa polypeptide is encoded by thechloroplast genome and synthesized in the organelle. Mutant plantsresistant to triazine herbicides have been obtained; they contain amutant 32 kDa protein in which the plastoquinone can no longer bedisplaced by triazine herbicides.

[0007] Other herbicides are known to block specific steps in amino acidsynthesis. For example the sulfonylureas are known to inhibitacetolactate synthase which is involved in isoleucine and valinesynthesis. Glyphosate inhibits the function of5-enol-pyruvyl-3-phospho-shikimate synthase, an enzyme involved in thesynthesis of aromatic amino acids. These enzymes are nuclear encoded butare translocated as precursers into the chloroplast.

[0008] Synthesis and import into the chloroplast of precursor proteinsare highly energy consuming processes. It would therefore be of interestto engineer foreign genes, particularly those which have products whichare functional within a chloroplast, through the chloroplast genomeinstead of the nuclear genome. By chloroplast is intended both matureand immature forms as well as organelles having substantially similarfunction in tissues other than leaf.

[0009] Relevant Literature

[0010] Uptake and expression of bacterial and cyanobacterial genes byisolated cucumber etioplasts (immature chloroplasts) has been described.Daniell and McFadden, Proc. Nat'l Acad. Sci. (USA) (1987) 84:6349-6353.Stable transformation of chloroplasts of Chlamydomonas reinhardtii (agreen alga) using bombardment of recipient cells with high-velocitytungsten microprojectiles coated with foreign DNA has been described.See, for example, Boynton, et al. Science (1988) 240:1534-1538; Blowers,et al. Plant Cell (1989) 1:123-132 and Debuchy et al., EMBO J. (1989)8:2803-2809. The transformation technique, using tungstenmicroprojectiles, is described by Kline et al. Nature (London)(1987)327:70-73. Manipulation of chloroplast genes has been described, forexample, generation of chloroplast mutants, Maliga et al., Nature (1975)255:401-402; protoplast fusion, Belliard et al., Mol. Gen. Genet (1978)165:231-237; organelle inactivation, Aviv et al., Plant Cell Rep. (1986)3:227-230; and chloroplast recombination, Medgyesy et al., Proc. Nat'lAcad. Sci. USA (1985) 82:6960-6964.

[0011] Tewari and coworkers have recently mapped two replication originsin pea cpDNA by electron microscopic analysis. Both of the origins ofreplication, identified as displacement loops (D loops), were found tobe highly active in DNA synthesis when used as templates in a partiallypurified replication system from pea chloroplasts (Cheung et al., supra,Merker et. al., Mol. Cell Biol. (1985) 8:1216-1223 and Boutry et al.Nature (London) (1987) 328:340-342).

[0012] Methods for targeting foreign gene products into hloroplasts(Shrier et al., EMBO J. (1985) 4:25-32) or itochnodria (Boutry et al.,supra) have been described. See also Tomai et al. Gen. Biol. Chem.(1988) 263:15104-15109 and U.S. Pat. No. 4,940,835 for the use oftransit peptides for translocating nuclear gene products into thechloroplast. Methods for directing the transport of proteins to thechloroplast are reviewed in Kenauf TIBTECH (1987) 5:40-47. Articlesrelating to herbicide tolerance and resistance include Shah et al.Science (1986) 233:478-481 and Mazur et al World Biotech (1985)2:97-108.

SUMMARY OF THE INVENTION

[0013] In accordance with the subject invention, methods andcompositions are provided for introducing heterologous DNA into thechloroplast genome. The method includes the steps of preparing anexpression cassette which includes a DNA fragment comprising asufficient portion of the 5′ untranslated region of a bacterial or achloroplast gene to provide for transcription and translation of thegene of interest, the gene of interest and a translational andtranscriptional termination region functional in the chloroplast. Theexpression cassette may optionally include DNA sequences which providefor integration of the gene of interest into the chloroplast genome,and/or an origin of replication capable of providing for replication ofa gene of interest in the chloroplast.

[0014] Preferred techniques for obtaining transformation of theexpression cassette into the chloroplast include bombardment ofchloroplast containing cells or tissue with high density metallicparticles to which the expression cassette has been absorbed. The methodfinds use in obtaining cells containing transgenic chloroplasts,including both dicotylenous and monocotylenous cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows the plasmid pHD407 which carri<a 4.1-kbp SmaIfragment insertion containing the orrigin of replication (D loop) frompea cpDNA inserted into pHD312. The plasmid pHD312 contains the entirepromoter and 5′ untranslated region of the pea psbA gene inserted 5′proximal to the promoterless cat gene present in the promoter selectionvector pkk232-8.

[0016]FIG. 2 shows alkaline agarose gell of in vitro repliationproducts. In vitro replication reactions were carried out as describedin ref. 20. After phenol extraction and ethanol precipitation, thesamples were separated in a 0.8% alkaline agarose gel, and the gel wasdried and autoradiographed. Phage λ DNA markers were idgested withHindIII and 5′ end-labeled with [α-³²P]ATP and polynucleotide kinase.

[0017]FIG. 3 shows (a) analysis of cat expression in tobacco NT1suspension cells bombarded with pUC118 (negative control), 35S-CAT(nuclear expression vector), and pHD407 (chloroplast expression vectorcontaining chloroplast replicon). The average protein concentrations in500 μl of the samples assayed are as follows. 48 hr: pUC118, 900 μg;35S-CAT, 476 μg; pHD407, 870 μg; 72 hr: pUC118, 787 μg; 35S-CAT, 590 μg;pHD407, 710 μg; 966 hr: pUC118, 360 μg; 35S-CAT, 147 μg; pHD407, 48 μg;120 hr: pUC118, 523 μg; 35S-CAT, 91 μg; pHD407, 99 μg. (b) Analysis ofcat expression in tobacco NT1 suspension cells bonbarded withchloroplast expression vectors containing various replicon fragments andpromoters. Protein concentration in samples assayed (72 hr afterbombardment) are as follows. (upper) pUC118: 856, 1008 μg; pHD312(repliconless): 802, 1075 μg; pHD407 (pea replicon): 1075, 488 μg;35S-CAT: 1160, 1102 μg. The film was exposed to the TLC plate for 8 hr.(Lower) pUC118; 65, 62 μg; pHD312:40, 46 μg; pHD407; 32, 26 μg; 35S-CAT;39, 38 μg; rbcL-CAT (tobacco replicon): 276 μg; rbcL-CAT (maizereplicon): 275 μg; rbcL-CAT (no replicon): 248 μg. The film was exposedto the TLC plate for 4 days due to the low number of bombarded cells.

[0018]FIG. 4 shows quantitative expression of cat in tobacco Ntlsuspension cells assayed with identical protein concentrations in sonicextracts. After autoradiography of the separated acetylatedchloramphenicol forms for 4 hr. spots were scraped and radioactivity wascounted. Silica gel alone had a background of 2203 cpm; the ranges ofcpm between different samples were as follows: pUC19, 2705-6556 cpm;35S-CAT, 3993-220, 353 cpm; pHD312, 2410-133, 240 cpm; pHD407,7484-267,364 cpm.

[0019]FIG. 5 shows kinetics of chloramphenicol acetylation with[¹⁴C]acetyl CoA, in tobacco NT1 protoplasts electroproated with foreignDNA. Pelleted protoplasts were resuspended in (400 μl) extraction buffer(5 mM EDTA/0.25 M Tris-HCl, pH 7.8, and 1.0 μg each anrtipain andpepstatin per ml), sonicated for 20 sec, and centrifuged in a Microfugefor 5 min at 4° C. to pellet the debris the extract (190 μl) after heattreatment (65° C., 10 min) was mixed with [¹⁴C]acetyl CoA (0.1 μCi; 1Ci=37 GBq) and chloramphenicol (40 μl of 8 mM stock). The reaction wascarried out at 37° C. using the highly quantitative two-phase assaysystem described by neumann et al (26). Slope values derived from datapoints and DNA concentrations used for eletroporation were as follows:pUC19, 31.0 (15 μg); pHD407, 24.2 (50 Ag); 35S-CAT, 114.1 (15 μg). Thecorrelation coefficient varied between 0.9 and 0.99 for different setsof experiments.

[0020]FIG. 6 shows Chloroplast Expression Vector pHD203. This vectorcontains a double psbA promoter inserted in opposite orientations tofaciliate insertion of additional genes. E. coli uidA gene coding forβ-glucuronidase has been inserted into the MCS.

[0021]FIG. 7 shows foreign gene expression studies in anther derivedgreen and albino plants. (a) Green and white albino wheat platnsregenerated from anther culture. (b) Expression of GUS in the albinowheat leaf bombarded with pHD203-GUS (top) and lack of expression in theleaf bombarded with pUC 19 (bottom). (c) Expression of GUS in the greenwheat leaf bombarded with pB1121 (middle), pHD 203-GUS (right); note thelack of GUS expression but the presence of tungsten particles in theplant bombarded with pUC19 (left). (d) Microscopic observation of GUSexpression in cells from a green leaf bombarded with pB1121. Note thepresence of GUS cleaved product spread evenly all around the cytosol.(e, f) Subcellular localization of GUS expression in chloroplasts ofcells from a green leaf combarded with pHD203-GUS. Note green, bluegreen and green plastids present inside the cells. (g, h) Foreign geneexpression studies in wheat callus derived from immature embryos: Calliwere bombarded with G) pUC19H) pHD203-GUS. Note tungsten particles atthe site of bombardment and the smaller blue spots scattered all aroundthe callus bombarded with pHD 203-GUS.

[0022]FIG. 8 shows expression of pHD203-GUS over time. Cells collectedon filter paper by vacuum filtration were transferred onto NT1 mediumsolidified with 0.2% geirite and bombarded with helium entrainmentconfiguration at 1500 psi, sample 8.1 cm from launch point using 1.0sleeve level. At each time point (days after bombardment), cells wereassayed for GUS activity. In both experiments, each treatment contained3 replicates.

[0023]FIG. 9 shows transient expression of GUS activity followingPEG-mediated protoplast transformation. In both experiments, eachtreatment contained 3 replicates.

[0024]FIG. 10 shows effect of osmoticum on chloroplast transformation.NTl cells collected on filter paper by vacuum filtration weretransferred onto 0.2% geirite solidified NTl media supplemented withdifferent concentrations of osmoticum (½ sorbitol and ½ mannitol),incubated for at least 1.5 hour and bombarded. After three days ofincubation, the cells were assayed for GUS activity. In experiment 1,each treatment had 5 replicates, and in experiment 2, each treatment had4 replicates.

BRIEF DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0025] Methods and compositions are provided for obtaining cellscontaining chloroplasts into which heterologous DNA has been inserted.The method includes the steps of preparing an expression cassette. Byexpression cassette is intended a DNA construct comprising a codingsequence and appropriate control sequences to provide for properexpression of the coding sequence in the chloroplast. The expressioncassette generally includes the following minimum components, the 5′untranslated region from a microorganism gene or chloroplast gene suchas psbA which will provide for transcription and translation of a DNAsequence encoding a polypeptide of interest in the chloroplast; a DNAsequence encoding a polypeptide of interest such as genes which providefor herbicide resistance or encode insecticidal proteins; and atranslational and transcriptional termination region such as a 2′inverted repeat region of a chloroplast gene that could stabilize RNA ofintroduced genes, thereby enhancing foreign gene expression. A host cellwhich contains chloroplasts is transformed with the expression cassetteand then the resulting cell containing the transformed chloroplasts isgrown to express the polypeptide of interest. The cassette mayoptionally include an antibiotic resistance gene in addition to amutated native chloroplast gene such as rbcL or 16SrRNA. In this option,expression of a desirable alteration of a native protein may be favoredin transformed chloroplasts by antibiotic selective pressure.

[0026] Typically, the expression cassette is flanked by convenientrestriction sites for insertion into an appropriate genome. Theexpression cassette may be flanked by DNA sequences obtainable fromchloroplast DNA which facilitate stable integration of the expressioncassette into the chloroplast genome, particularly by homologousrecombination. Alternatively, the expression cassette may remainunintegrated by including an origin of replication such as is obtainablefrom chloroplasts which is capable of providing for replication of theheterologous DNA in the chloroplast.

[0027] Plants containing these cells can be generated and grown toproduce seed. The system finds use in the production of cells and plantswherein polypeptides of interest can be expressed directly in thechloroplast, for example, to provide for herbicide resistance.

[0028] Transformation of the chloroplast genome as opposed to thenuclear genome has several advantages. For example, chloroplast genes ingeneral are maternally inherited. It therefore may be safer to releasechloroplast transgenic plants into the environment since the possibilityof “escapes” of foreign genes through dispersal of pollen grains iseliminated. Further, it is possible to introduce multiple copies offoreign genes into the chloroplast genome as opposed to the limitednumber of functional copies of a foreign gene which typically may beintroduced via the nuclear genome. Plants engineered through thechloroplast genome rather than the nuclear genome also could have asignificant energy advantage since synthesis and import of precursorproteins into a cell organelle are highly energy consuming and ratelimiting processes.

[0029] In preparing the expression cassette, the various DNA sequencesmay normally be inserted or substituted into a bacterial plasmid. Anyconvenient plasmid may be employed, which will be characterized byhaving a bacterial replication system, a marker which allows forselection of the bacterium, and generally one or more unique,conveniently located restriction sites. The plasmids, or vectors, mayinclude such vectors as pUC, pBR322, pBlueScript, and pGEM, theparticular plasmid being chosen based on the nature of the markers,availability of convenient restriction sites, copy number and the like.

[0030] A strategy which allows for the stepwise combination of thedifferent fragments is then defined. As necessary, the fragments may bemodified by employing synthetic adapters, adding linkers, employing invitro mutagenesis or primer repair to introduce specific changes in thesequence, which may allow for the introduction of a desired restrictionsite or a suitably altered biological activity for removing superfluousbase pairs or the like. By appropriate strategies, the number ofmanipulations required as well as the degree of selection required ateach stage of manipulation can be minimized. After each manipulation,the vector containing the manipulated DNA can be cloned, the clonescontaining the desired sequences isolated, and the vector isolated andpurified. As appropriate, hybridization, restriction mapping orsequencing may be employed at each stage to insure the integrity andcorrectness of the sequence.

[0031] For transcription and translation of the DNA sequence encoding apolypeptide of interest, the entire promoter region from a gene capableof expression in the chloroplast generally is used. The promoter regionmay include promoters obtainable from chloroplast genes, such as thepsbA gene from spinach or pea, the rbcL and atpB promoter region frommaize and rRNA promoters. Examples of promoters are described inHanley-Bowdoin and Chua, TIBS (1987) 12:67-70; Mullet et al., PlantMolec. Biol. (1985) 4:39-54; Hanley-Bowdoin (1986) PhD. Dissertation,The Rockefeller University; Krebbers et al., Nucleic Acids Res. (1982)10:4985-5002; Zurawski et al., Nucleic Acids Res. (1981) 9:3251-3270;and Zurawski et al., Proc. Nat'l Acad. Sci. U.S.A. (1982) 79:7699-7703.Other promoters may be identified and the relative strength of promotersso idenntified evaluated, by placing a promoter of interest 5′ to apromoterless marker gene and observing its effectiveness relative totranscription obtained from, for example, the promoter from the psbAgene, the strongest chloroplast promoter identified to date. Theefficiency of foreign gene expression additionally may be enhanced by avariety of techniques. These include the use of multiple promotersinserted in tandem 5′ to the DNA sequence of interest, for example adouble psbA promoter, the addition of enhancer sequences and the like.

[0032] For the most part, promoters functional in the chloroplast areconstitutive rather than inducible. However, where it is desired toprovide for inducible expression of the polypeptide of interest, aregulatable promoter and/or a 5′ untranslated region containingsequences which provide for regulation at the level of transcriptionand/or translation (at 3′ end) may be provided. Transcription and RNAstability appear to be important determinants of chloroplast geneexpression. For example, the 5′ untranslated region may be used from agene wherein expression is regulatable by light. Similarly, 3′ invertedrepeat regions could be used to stabilize RNA of foreign genes.Regulatable genes may be identified by enhanced expression in responseto a particular stimulus of interest and low or absent expression in theabsence of the stimulus. For example, a light regulatable gene may beidentified where enhanced expression occurs during irradiation withlight, while substantially reduced expression or no expression occurs inthe negligible of light.

[0033] The DNA sequence encoding the polypeptide of interest may be astructural gene or a portion thereof which provides for a desiredexpression product. The gene may be any gene, whether native, mutantnative, or foreign to the plant host. By foreign is intended a gene notendogenous to the host cell, but may include indigenous sequences, suchas viral sequences and bacterial sequences which are naturallyassociated with the plant cell.

[0034] For native and mutant native genes, the gene may provide forincreased capability of amino acid synthesis, enhanced response tolight, herbicide resistance, for example, to glyphosate or atriazine,improved drought resistance, insect resistance (BT toxin gene), alteredfatty acid synthesis, such as an increase in unsaturated fatty acids,enhanced fixation of CO₂. Foreign genes may include enhancement ofnative capabilities, herbicide resistance, resistance to various pests,such as viruses, insects, bacteria or fungi, production of foreignproducts, as a result of expression of one or more foreign genes, or thelike. Mutant native genes include 16SrRNA or its renal RNA gene, thealtered native gene provides resistance to antibiotics, and a mutantrbcL (ribulose biphosphate carbonylase/oxygenase, large subunit. Thealtered native rcbL gene encodes a more active carbonylase which canenhance plant productivity.

[0035] In many instances, it will be desirable to have at least oneadditional structural gene such as a gene providing for antibioticresistance or functional portion thereof to serve as a marker associatedwith the expression cassette. Those plant cells in which the foreigngene has been stably introduced can be detected by means of the markergene. Of course, one may provide for a string of expression constructshaving a plurality of the same or different genes in the construct. Thusthe presence of only two genes is merely illustrative. As markers, forstructural genes, one can employ β-lactamase, herbicide resistance genessuch as a mutant psbA gene or EPSPS-aroA. Other markers can include thecat gene, which encodes chloramphenicol acetotransferase and the uidagene which encodes β-glucuronidase (aus).

[0036] The DNA sequence encoding the polypeptide of interest may besynthetic, naturally derived, or a combination thereof. Depending uponthe nature of the DNA sequence of interest, it may be desirable tosynthesize the sequence with chloroplast preferred codons. Thechloroplast preferred codons may be determined from the codons ofhighest frequency in the proteins expressed in the largest amount in theparticular plant species of interest. Depending upon the desiredapplicability of transforming the chloroplast genome, the DNA sequencemay be inserted relative to the promoter in either the sense or theantisense direction.

[0037] The termination region which is employed will be primarily one ofconvenience, since the termination region appears to be relativelyinterchangeable among chloroplasts and bacteria. The termination regionmay be native to the transcriptional initiation region, may be native tothe DNA sequence of interest, or may be obtainable from another source.Convenient termination regions are available from. (See, for example,Chen and Orozco, Nucleic Acids Res. (1988) 16:8411).

[0038] The transcription level in the chloroplast should be sufficientto provide sufficient RNA capable of resulting in a modifiedchloroplast. A “modified chloroplast” is a chloroplast having adetectably different phenotype from a chloroplast of the same specieswhich has not been transformed or expressing a foreign gene product,that is, one not containing the expression cassette in question. Variouschanges in phenotype are of interest and include modification oralteration of native capabilities (either enhancement or diminution),absence of a native capability (for example, as the result-oftransformation using antisense sequences) or addition of a newcapability. Examples include a chloroplast containing a mutant psbA geneand as a result capable of resistance to the herbicide atrazine or achloroplast containing a foreign gene product such as EPSP synthase andthereby capable of resistance to the herbicide glyphosate or expressinga BT toxin gene and capable of resistance to worms and insects.

[0039] Where it is desired to obtain replication of a plasmid containingthe expression cassette in the chloroplast any chloroplast origin ofreplication may be used in the expression construct.

[0040] The expression cassettes may be transformed into a plant cell ofinterest by any of a number of methods. These methods include, forexample, biolistic devices (See, for example, Sanford, Trends InBiotech. (1988) 6:299-302, U.S. Pat. No. 4,945,050; electroporation(Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985) 82:5824-5828); useof a laser beam, electroporation, microinjection or any other methodcapable of introducing DNA into a chloroplast. The use of thesetechniques permits the application of the invention described herein ina wide variety of both monocotyledonous and dicotyledonous plant cells.

[0041] For use in the bombardment transformation technique, anexpression cassette is adsorbed to a bombardment particle, typicallyconsisting of tungsten particle having an average size of about 0.7 μm.Particles consisting of other metals having a density similar totungsten may also find use, such as gold, platinum and the like.Typically, about 2-5 μg of DNA is adsorbed per pg of tungsten, usuallyabout 2 μg of DNA per μg of tungsten. Following adsorption of the DNA tothe particles, any clumps of particles are dispersed, for example, bysonication. Any method to fix the DNA on the outside surface of themetal bombardment particles is acceptable and are known to those skilledin the art. (See, for example, Sanford et al. (1988) supra and Klein etal. supra). The DNA must be secured to the particles for delivery, butnot fixed in such a manner as to impede release of the DNA into thecell.

[0042] For transformation, about 100-500 μg, generally approximately 200μg of bombardment particles to which the expression cassette has beenadsorbed are loaded into a particle gun (such as those available fromBiolistics, Inc. and DuPont) according to the manufacturer's directions.Isolated cells generally 100-300 μg fresh weight per petri plate (5 cmdiameter) are placed on a growth surface, such as a petri dish or tissueculture dish containing for example Whatman #1 filter paper with growthmedium to adhere the cells to a solid surface. The cells do not have tobe in a single cell layer, but may be a few layers thick. Theimmobilized cells are placed in the bombardment chamber of the particlegun and placed under as high a vacuum as feasible, generally about 0.07to 0.3 atmospheres, preferably 0.07. The pressure in the sample chamberis then reduced to about 0.07 atmospheres prior to bombardment. Thecells preferably are bombarded about 10 cm from the end of the barrel ofthe particle gun (at the fourth level in the DuPont gene gun). Thefiring mechanism of the particle gun is activated and the cells arebombarded from 1-3 times, generally twice to increase the number oftransformed cells. The vacuum is then released, and the bombarded cellsplaced in fresh growth medium at about 26° C., preferably in the lightin growth chambers. Other bolistic devices include the use of “flyingdiscs” such as those made from plastic membranes or discs made of, forexample nylon mesh (94 μm) (““helium entrainment” method).

[0043] A plant containing transgenic chloroplasts may be generated whenthe host cell used in the transformation process possesses totipotency.Procedures for regeneration of transgenic plants from transformed cellsor tissues are, in general, similar, with suitable modifications withinthe capability of one skilled in the art. Regeneration of dic such assugar beets, Freytag et al. Plant Cell Rep. (1988) 7:30-34; tobacco,Svab et al. Proc. Nat'l Acad. Sci. U.S.A. (1990) 8526-8530 or monocotssuch as wheat from anthers or embryos (see below) routinely has beensuccessful.

[0044] If it is desired to transform chloroplasts in other plantmaterials, for example anther culture derived plants or embryo derivedcallus, the plant material is placed in a convenient container, forexample a petri dish as described above for isolated cells.

[0045] The presence of the desired gene in the plant chloroplast can beestablished in a variety of ways, depending upon the nature of the gene.Techniques such as the Northern blot can be employed for detectingmessenger RNA which codes for the polypeptide of interest. In addition,the presence of expression can be detected in a variety of ways. Wherethe expression product provides a detectable phenotype, such as a novelphenotype or modification of an endogenous trait, the expression of thedesired product may be determined by detecting the phenotype. Where adetectable phenotype is not available, antibodies specific for themature product may be employed. The chloroplasts may be isolated inaccordance with conventional ways, disrupted and the western or othertechnique employed to identify the presence of a desired product. Thepresence of a gene which produces an exogenous product may be detectedby isolation and lysis of the chloroplast. The resulting cellularmaterial may then be assayed for the exogenous product or the exogenousgene. The exogenous product may be detected by electrophoresis,chroma-tography, immunoassay or the like. The gene may be detected forexample by hybridization using Southern blotting. The transientexpression system, reported here, should facilitate studies on foreigngene expression, regulation, or DNA replication in plastids in vivo.Thus, an approach may be opened to major advances in genetic engineeringof higher plant organelles.

[0046] Once the chloroplast has been shown to have been transformed, thecells of the plant may be used repeatedly for tissue culture, followedby a growth of callus tissue were desired or regeneration of a plant.Thus, the modified plant cell may be repetitively regenerated by use ofcell and tissue culture. In some instances, proper propagation may bemaintained from seed.

[0047] As a host cell, any of a number of plant cells may be employedsuch as sugar beet, tobacco, wheat, etc.; plant parts of interestinclude leaves (chloroplasts), flowers (chromoplasts), roots, such astubers (amyloplasts); fruits (chromoplasts); sprouts (germinatingseedlings), etioplasts); sea weeds/algae (chloro/chromoplasts).

[0048] Constructs containing a selectable marker when expressed in thechloroplasts (for example resistance to a herbicide such as glyphosateor atrazine) may when used in conjunction with another structural gene,be used to select for chloroplasts, cells or plants containing theconstructs.

[0049] The described methods and compositions find particular use inproviding an altered phenotype in plant chloroplasts, such as providingdirectly for herbicide resistance and the like. The constructs mayprovide a means for selecting cells and plants containing transformedchloroplasts, where a selectable chloroplast function is included in theconstruct.

[0050] The following examples are offered by way of illustration and notby limitation.

EXPERIMENTAL EXAMPLE I Transient Foreign Gene Expression in Chloroplastsof Cultured Tobacco Cells After Bolistic Delivery of Chloroplast Vectors

[0051] Construction of Chloroplast Expression Vectors.

[0052] A series of chloroplast expression vectors has been constructedusing the promoter selection vector pKK232-8 (Pharmacia), which is apBR322 derivative containing a promoterless cat gene. Restrictionfragments of chloroplast DNA (cpDNA) containing the entire promoterregion and 5′ untranslated region of the psbA gene from spinach (pMP450,a gift from Wilhelm Gruissem, University of California, Berkeley) pHD306or pea (pPPBX10218, a gift from John Mullet, Texas A & M University)pHD312 or, alternatively, the rbcL and atpB promoter region from maize(pPBI1443, a gift from Antony Gatenby, E.I. DuPont de Nemours & Co.,Wilmington, Del.) pHD103 have been individually inserted into themultiple cloning site (MCS) that exists 5′ proximal to the promoterlesscat gene. The strength of each promoter; has been investigated byanalyzing transient expression of cat in cucumber etioplasts, asdescribed in Daniell and McFadden, Proc. Nat'l Acad. Sci. U.S.A. (1987)84:6349-6353.

[0053] To study cat expression in the cytosol, a 35S-CAT constructobtained from Abdul Chaudhury (Ethan Signer's laboratory, MIT) has beenused. This is a 4.2-kilobase-pair (kbp) plasmid designatedpUC8CaMVCATψN, with a cat gene driven by a 35S cauliflower mosaic viruspromoter and flanked by a 3′ Nos PstI fragment. For negative controls,pUC118 or pUC19 has been used in all experiments.

[0054] In Vitro Replication Studies.

[0055] A replication fraction containing RNA polymerase, DNA polymerase,DNA primase, and topoisomerase I activities was isolated from peachloroplasts as described by Meeker et al. (Molec. Cell. Biol. (1988)8:1216-1223). The heparin-Sepharose fraction was used for in vitroreplication reactions. Insertions of a variety of replicon fragmentsinto chloroplast expression vectors is described below.

[0056] Bombardment of Suspension Cells with Microprojectiles.

[0057] To prepare tobacco NT1 cells for bombardment withmicroprojectiles, about 100 mg of 4-day-old suspension cells (freshweight) was collected on filter paper (Whatman no. 1.5.5 cm) by vacuumfiltration of 5 mlof suspension culture (1×10⁶ cells per ml). Two layersof filter paper were placed inside a 5.5-cm Petri dish and moistenedwith 1.4 ml of MS medium (Murashige and Skoog Physiol. Plant. (1962)15:473⁻⁴97). The single filter paper bearing the cells was then placedover the two layers of filter paper.

[0058] Tungsten particles coated with DNA were prepared essentially asdescribed by Boynton et al. (Science (1988) 240:1534-1538). To adsorbDNA to the microprojectiles, 2.5 μl of DNA (1 μg/pg of TE buffercontaining 10 mM Tris HCl and 1 mM EDTA, pH 7.7) was added to 25 μl of asuspension of tungsten particles (0.05 mg/ml of 50% glycerol) in a1.5-ml Eppendorf tube. After addition of the DNA, CaCl₂ (25 μl of a 2.5M solution) and spermidine free base (5 μl of a 1 M solution) were addedto the suspension. After 10 min of incubation, the particles werepelleted by centrifugation in a Microfuge for 30 sec. and a portion ofthe supernatant (25 μl) was removed. The final microprojectilepreparation therefore contained 39 μg of tungsten per p1 of suspensionand 2 μl of DNA per p1 of tungsten. The clumps of particles weredispersed by briefly (1 sec) touching the outside of the Eppendorf tubeto the probe of a sonicator. After sonication, 5 μl of the tungsten/DNAsuspension was placed on the front surface of a cylindrically shapedpolyethylene macroprojectile. The macroprojectile was then placed intothe barrel of the particle gun and accelerated.

[0059] Cells were bombarded 10 cm from the end of the barrel of theparticle gun. The pressure in the sample chamber was reduced to 0.1atmosphere prior to bombardment. In all experiments, three replicatePetri plates were bombarded per treatment. After the addition of freshgrowth medium, the cells were maintained at 25° C. in the light, inplant growth chambers.

[0060] Analysis of Cat Expression in NT1 Cells and Protoplasts.

[0061] Cultured tobacco cells were transferred to Corex tubes and washedonce with 10 ml of TE buffer containing 250 mM Tris HCl and 10 mM EDTA(pH 7.8). Cells centrifuged at 8000×g for 10 min were transferred to2-ml Eppendorf tubes and resuspended in 1 ml of TE buffer (pH 7.8)containing 2 mM phenylmethylsulfonyl fluoride. The cells were sonicatedtwice for 20 sec each, using a probe sonicator. After a 15-mincentrifugation at 4° C., the supernatants were transferred to newEppendorf tubes and assayed for cat activity as described in Daniell andMcFadden, supra. NT1 protoplasts were prepared and electroporated asdescribed by Paszty and Lurquin (Biotechniques (1987) 5:716-718) exceptthat the 250 μF capacitor was charged to 250 V and 1.5 millionprotoplasts were used per electroporation event.

[0062] Expression of Foreign Genes in Isolated Chloroplasts

[0063] The cucumber etioplast in organello transient expression system(See Daniell and McFadden, supra) was used to test the strength ofchloroplast promoter fragments inserted into the MCS 5′ proximal to thepromoterless cat gene-(FIG. 1). Transient expression of cat inEDTA-washed etioplasts isolated from hormone-pretreated cucumbercotyledons revealed spinach or pea psbA promoter (pHD306, pHD312) to bethe strongest among the promoter fragments tested. Since expressionstudies after bombardment of foreign DNA were planned to be conducted innonphotosynthetic cells, the transient expression system in etioplastsserved as an analogous comparable system to the plastids present incultured tobacco cells.

[0064] In Vitro Replication of Chloroplast Expression Vectors.

[0065] Plasmid construction pCB1-12 is a 10-kbp BamHI pea cpDNA fragmentin pBR322, containing both D-loop regions (See Tewari et al., supra).The plasmid pCPH5.6 is a 5.6-kbp PstI-HindIII cpDNA fragment in pUC19containing one of the two D-loop regions. The plasmid pCE2.1 is a2.1-kbp EcoRI cpDNA fragment from the region between the two replicationorigins. Plasmid pCP1.8 is a 1.8-kbp PsiI clone from a distant region ofthe cpDNA. All of these constructions were used as templates for invitro DNA synthesis using a replication fraction isolated from peachloroplasts, and the in vitro replication products were analyzed onalkaline agarose denaturing gels. As seen in FIG. 2, the autoradiogramshowed lack of synthesis of any full-size DNA molecule in pCP1.8 andpCE2.1, which do not contain a D-loop region; the autoradiogram clearlyshowed in vitro synthesis of single-stranded DNA molecules of about 14.5and 6.3 kbp. corresponding to full-length pCB1-12 and pCS4.1,respectively. The 4.1-kbp SmaI fragment in pCS4.1, found to be active inin vitro DNA synthesis, was inserted into the chloroplast expressionvector pHD312, which contains the pea psbA promoter 5′ proximal to thecat gene. The resultant construction, pHD407, when analyzed for in vitroDNA synthesis, appeared as a single-stranded DNA molecule of about 9.9kbp in an alkaline agarose denaturing gel (FIG. 2).

[0066] Expression of Cat in Tobacco NT1 Suspension Cells.

[0067] Expression of cat was assayed in sonic extracts incubated in thepresence of (dichloroacetyl-1-¹⁴C)chloramphenicol and acetyl coenzyme.Sonic extracts of cells bombarded with pUC118 showed no detectable catactivity in the autoradiograms; cells bombarded with 35S-CAT showedmaximal expression 72 hr after bombardment, whereas those bombarded withpHD407 and pHD312 showed a low level of expression until 48 hr ofincubation (FIG. 3A). A dramatic increase in the expression of cat wasobserved 24 hr after the addition of fresh medium to cultured cells insamples bombarded with pHD407.

[0068] The repliconless chloroplast expression vector pHD312 showedmaximal activity at 72 hr of incubation, which is about 50% of theactivity observed with pHD407 at that time point (FIG. 4). A high levelof cat expression was maintained with subsequent incubation of culturedcells bombarded with pHD407, whereas the expression of nuclear cat andthe repliconless chloroplast vector sharply declined. Quantitativestudies confirmed the earlier visual observation (FIG. 4). These resultssuggest that the “biolistic” process delivers foreign DNA intochloroplasts of cultured tobacco cells and that the marker gene isexpressed in appropriate compartments. The kinetics of cat activity, asa function of incubation time after bombardment, may be interpreted asan indication that the delivery process into the chloroplast is not asefficient as into the nucleus since expression of all chloroplastvectors is delayed until 72 hr. Chloroplast vectors with the replicon(pHD407) may subsequently replicate autonomously inside thechloroplasts, resulting in a higher level of expression. However, thecontribution of the upstream promoter sequences of 23S rRNA gene,present in the replicon fragment in pHD407, to enhancedtranscription/translation of the cat gene cannot be ruled out.

[0069] These observations were subsequently confirmed by investigationsusing similar chloroplast expression vectors provided by L. Bogorad'slaboratory. Expression of cat was studied in NT1 cells bombarded withvectors containing replicon inserts from tobacco and maize chloroplastgenomes (FIG. 3B). A tobacco Bam4 cpDNA fragment was cloned into pGV825(a Ti plasmid intermediate vector) to produce pACpl8; this fragmentcloned into pUC supports DNA synthesis in vitro using the replicationsystem described by Carrillo and Bogorad (Nucleic Acids Res. (1988)16:5603-5620). Maize Bam10 fragment was cloned into pGV825 to producepACp19; this fragment cloned into pBR322 is not especially active in thein vitro DNA synthesis assay of Tewari and co-workers (Proc. Nat'l Acad.Sci. U.S.A. (1987) 84:194-198) but functions as an autonomouslyreplicating sequence in yeast (when cloned into YPI5). The repliconlessvector showed 0.74×10³ cpm cat activity per μg of protein in sonicextract of cells 72 hr after bombardment; vectors containing repliconfragments from tobacco and maize showed 1.03 and 1.45×10³ cpm per μg ofprotein, respectively. In all of these constructs, the bacterial catgene is under the control of an rbcL promoter region from maize.

[0070] Sterility tests to ensure absence of microbial contamination ofthe cultures were performed by streaking an aliquot of cells orprotoplasts on LB agar plates, for every time point, after bombardmentor prior to sonication. Among the cat assays presented in FIG. 3A, twocontaminants were noticed. These samples were discarded and hence fewercat assays have been presented for the last time point. No bacterialcontamination has been observed in other batches of bombarded cells.Expression studies have been performed in six different batches ofcells, varying several parameters.

[0071] Organelle-specific expression of cat in appropriate compartmentswas checked by introducing all three plasmid constructions, pUC19,35S-CAT, and pHD407, into tobacco protoplasts by electroporation (FIG.5) and assaying using the highly quantitative two-phase assay system(Neumann et al., Biotechniques (1987) 5:444-447). Although 35S-CATshowed expression of cat, no activity was observed with pUC19 andpHD407, establishing the fact that these two constructions do notexpress in the nucleus. It is known that electroporation of protoplastsdelivers DNA into the cytosol but not inside organelles. Furthermore,recent attempts to induce tobacco psbA promoter to function in thenuclear compartment (to study transient expression of bar or nptIIgenes) revealed the absolute need to insert 35S promoter or enhancerelements 5′ proximal to the psbA promoter region (Cornelissen andVandewiele, Nucleic Acids Res. (1989) 17:19-29). Bogorad and co-workersalso observed that chloroplast genes were not transcribed from their ownpromoters when placed in the nuclei of transgenic tobacco plants (Cheunget al., Proc. Nat'l Acad. Sci. U.S.A. (1988) 85:391-395).

EXAMPLE II Transient Foreign Gene Expression in Different CellularComponents of Wheat Leaves and Calli After Bolistic Delivery

[0072] Construction of the Chloroplast Expression Vector, pHD203-GUS

[0073] The chloroplast expression vector pHD203 (FIG. 6), a pBR322derivative, contains a double psbA promoter fragment (from the peachloroplast genome) inserted in opposite orientations to facilitatesimultaneous transcription of two promoterless genes. One of the twopsbA promoter regions drives the cat gene. The presence of ribosomal RNAT1 and T2 terminators distal to the cat gene aids transcriptiontermination, while the presence of three stop codons between the psbApromoter and the AUG of the cat gene prevents translational readthroughinto the cat gene. The second psbA promoter fragment has been placedupstream of a multiple cloning site (MCS) containing sites for EcoRI,AvaI, XmaI, SmaI, BamHI, SalI, HincII, PstI and HindIII. There is aribosomal RNA Ti terminator distal to the MCS to aid transcriptiontermination. There are convenient EcoRI and PstI sites within the catand β-lactamase genes, respectively, to screen for partial digestion ofpHD203. The E. coli uidA gene coding for β-glucuronidase (GUS)(Jefferson et al., Proc. Nat'l Acad. Sci. U.S.A. (1986) 83:8447-8451)was inserted into the MCS of pHD203 (FIG. 6.). The following restrictionsites originally present in pHD203 have been lost in pHD203-GUS: HindIII(208 in PHD203), HincII (196), SalI (194), BamHI (188) and SmaI (185).Restriction analyses were done to confirm proper construction beforeproceeding with studies on the expression of GUS.

[0074] Nuclear Expression Vector pBl121

[0075] The nuclear expression vector pBl121 (Dr. J. C. Sanford) carriesa GUS gene driven by a CaMV-35S promoter and flanked at the 3′ end by anos terminator and a polyA tail (Jefferson, Plant Mol. Biol. Rep. (1987)5:387-405). For negative control, pUC19 DNA was routinely used in allbombardments.

[0076] Plant Materials

[0077] Green and albino plants were obtained from anther culture (Zhouand Konzak, Crop Sci. (1989) 29:817-821) of wheat cultivars ‘Edwall’,Pavon 76‘and ’ Pavon 808′. When the plants were at the 2-3 leaf stage,they were transplanted aseptically into test tubes containing halfstrength MS media (Murashige and Skoog, (1962) supra, supplemented with2% sucrose and solidified with 0.8% agar. Calli were regenerated fromimmature embryos of wheat by a modified method of Sears and Deckard CropSci. (1982) 22:546-551. The 10-12 days old immature embryos were placedon basal MS media containing only half the amount of 2-4D (1 mg/l); thecalli were maintained in this medium until hard white embryogenic tissuedeveloped. This tissue usually starts forming after about 2 months whenthe calli are transferred to fresh media every 3-4 weeks. Calli rich inthis tissue were transferred to fresh media and used for bombardment.

[0078] Bombardment of Wheat Leaves or Calli With Micro-Projectiles:

[0079] Tungsten particles (60 mg, M-10) were soaked overnight in 1 ml-ofabsolute ethanol, after vortexing for 2 min. After washing thrice withautoclaved water, the particles were resuspended in 50% glycerol(sterile). Particles for bombardment were prepared as follows: 25 ul ofwashed particles was added to 5 ug of DNA in TE (10 mM Tris, 1 mM EDTA,pH 8.0) buffer followed by the addition of 25 ul of CaCl₂ (2.5M) and 5ul of spermidine (1M). After incubating the DNA with the tungstenparticles for 10 min at room temperature, 40 ul of the supernatant wasdiscarded after a brief centrifugation. Five ul of the remaining DNAcoated tungsten particles was loaded onto the macroprojectile. Prior tobombardment, anther culture-derived green or albino plants or embryoderived calli were placed in petri dishes (10 cm in diameter) in MSsalts (36) containing 2% sucrose, solidified with 0.8% agar. Keeping thesamples at the fourth level in DuPont's gene gun PDS-1000 and vacuum at0.07 atmosphere, each sample was bombarded twice. In all experiments,triplicate samples were bombarded per treatment. After bombardment, theplant materials were incubated at 26° C. for 72 hrs in growth chambers.Contamination by bacteria was continuously monitored by checking tissuesat various stages of the experiment and by bombarding plasmids that donot contain the GUS gene.

[0080] β-Glucuronidase Assay

[0081] Three days after bombardment, the tissues were assayed for GUSactivity by adding suitable aliquots of the GUS substrate that contained0.5 mg/ml 5-bromo-4 chloro-3-indoyl-B-D-glucuronic acid, and 0.1% ofTriton X-100 10 mM EDTA, 0.5 mM potassium ferrocyanide in 100% M sodiumphosphate buffer, pH 7.0. The Petri dishes were then sealed withParafilm and incubated at 37° C., overnight; cells or tissues wereobserved under the microscope and photographed.

[0082] GUS Expression in Anther Derived Albino Plants:

[0083]FIG. 7A shows the GUS was expressed in the albino leaf bombardedwith pHD203-GUS (left) but not in that bombarded with pUC19 (right). Theproduct of uida gene, β-glucuronidase, when present, cleavesβ-glucuronic acid from the substrate X-gluc(5-bromo-4-chloro-3-indoyl-B-D-glucuronic acid) to produce an insolubleindigo dye following oxidative dimerization. Even though some of theearlier investigations on pollen derived albino rice plants indicatedlack of ribosomes in albino plastids as the cause of albinism (Sun etal., Theor. Appl. Genet. (1979) 55:193-197, subsequent studies in otherlaboratories observed alterations in the albino plastid genome (Day andEllis, Cell (1984 39:359-368, and Current Genet. (1985) 9:671-678. Ourrecent studies have unequivocally established the presence of afunctional transcriptional/translational machinery in the plastids ofanther culture derived albino wheat plants (data not shown). Expressionof GUS in albino leaves bombarded with pHD203-GUS (FIG. 7A) furtherconfirms observations of the presence of a functional protein syntheticmachinery in albino plastids. Chloroplast specific expression of GUS bypHD203-GUS is discussed below in the section on “compartmentalized GUSexpression”.

[0084] GUS Expression in Anther Derived Green Plants:

[0085] Green plants derived from anther culture were preferred forstudies on gene expression because the results are comparable to fieldgrown plants but at the same time are free of bacteria since they hadbeen grown under totally sterile conditions. Though the tungstenparticles are seen in the sample bombarded with pUC 19 no GUS expressionis observed. On the other hand, it is evident from samples bombardedwith pBI121 and pHD203 that B-glucuronidase, when present, cleavedglucuronic acid from the substrate X-gluc to produce an insoluble indigodye.

[0086] Compartmentalized GUS Expression:

[0087] In order to identify the precise compartment in which pBI121 orpHD203-GUS function, bombarded leaves from anther derived green plantswere examined under the microscope. As shown in FIG. 7B, B-glucuronidasecleaved product, the indigo dye, was present evenly all around thecytosol, when nuclear expression vector pBI121 was bombarded into wheatleaves. When chloroplast expression vector pHD203-GUS was used forbombardment, the indigo dye was subcellularly localized within thechloroplasts of wheat cells (FIGS. 7C, D).

[0088] Chloroplast specific expression of pHD203-GUS has been confirmedby its failure to express when introduced into the cytosol ofprotoplasts by PEG-mediated transformation and distinctly differentkinetics of expression than the nuclear vector and subcellularlocalization of the cleaved product in cultured NT1 tobacco cells (datanot shown). These results also confirm earlier observations of thefailure of CAT expression in tobacco NT1 protoplasts uponelectroporation of chloroplast vectors containing the cat gene driven bya psbA promoter (Daniell et al., Proc. Nat'l Acad. Sci. U.S.A. (199087:88-92). More recently, the precise localization of chloroplastvectors inside plastids of cultured sugarbeets cells following biolisticdelivery, using an in vivo DNA replication system (see above) has beendemonstrated.

[0089] GUS Expression in Callus Derived from Immature Embryos:

[0090] While anther derived albino and green plants are ideal to studytransient expression of foreign genes, regeneration of wheat plants fromthem may be a formidable challenge. Therefore calli rich in embryonictissue were regenerated from immature embryos of wheat. FIG. 7E showsthe expression of GUS in regenerable callus derived from immatureembryos. When bombarded with foreign DNA, callus clumps were shatteredupon impact from the tungsten particles; however, this did not affecttheir subsequent gene expression. No background indigo dye was detectedin negative controls, bombarded with pUC19, when GUS substrate was added(FIG. 7F). Both large and small blue spots were observed in the callusbombarded with pHD203-GUS (FIG. 7F), indicating that chloroplasts in anumber of targeted cells have been transformed.

[0091] Transient expression of GUS in different cellular compartmentsfollowing biolistic delivery of chloroplast or nuclear expressionvectors into wheat cells, leaves or calli, derived from anther cultureor immature embryos, is reported here. Expression of GUS in albinoplastids when albino leaves were bombarded with pHD203-GUS confirms thepresence of a functional protein synthetic machinery in theseorganelles. When pBI121, the nuclear GUS vector was bombarded, theB-glucuronidase cleaved product was observed evenly all around thecytosol. When pHD203-GUS was bombarded, the indigo dye was subcellularlylocalized within the chloroplasts of wheat cells. GUS expression couldalso be observed in regenerable calli derived from wheat immatureembryos. Lack of expression of GUS in negative controls bombarded withpUC19 was evident.

EXAMPLE III Optimization of Delivery of Foreign DNA Into Higher PlantChloroplasts

[0092] New Biolistic Device

[0093] The standard gun-powder driven PDS-1000 biolistic device (DuPontCo.) was compared to a newly designed helium driven device. Briefly, asmall chamber is sealed at one end with one or more layers of rupturablemembrane (2 mil plastic membrane). The chamber is then filled to highpressure with helium gas. A solenoid-driven lance then ruptures themembrane, which releases a sharply defined shock wave. The shock wavethen enters a throat region of the device which contains a removablesleeve. Inside the sleeve are removable rings which are used to retainvarious microprojectile launching mechanisms. The principal mechanismemployed in this paper is helium entrainment. In the helium entrainmentconfiguration, a nylon mesh is locked into place across the axis of thesleeve. Microprojectiles (in suspension) are loaded directly onto thecenter of the mesh, and the helium shock wave atomizes and acceleratesthe microprojectiles as it passes through the mesh. A second mechanismemployed involves acceleration of a flying disc. In this configuration,a plastic membrane is loosely held in the same position as the nylonmesh. Particles are dried onto its surface and upon firing, the disc isaccelerated down the sleeve 1 cm, where it impacts against a screenstopping surface. The entire system is enclosed within a vacuum chamber.

[0094] Bombardment of Suspension Cells with Microprojectiles

[0095] To prepare tobacco NT1 cells (Paszty and Lurquin Paszty, supra)for bombardment with microprojectiles, about 100 mg of cells-from a4-day-old suspension were collected on filter paper (Whatman No. 1, 5.5cm) by vacuum filtration of 5 mls of the suspension culture. Two layersof filter paper were placed inside a 60-mm petri dish and moistened with1.5 ml of NTI medium (Paszty and Lurquin, supra. The single filter paperbearing the cells was then placed over the two layers of filter paper.The samples were bombarded with tungsten particles coated with DNA.

[0096] The DNA coating procedure was as described by Klein et al. exceptthat after 10 min. of incubation of the DNA-tungsten suspension, theparticles were pelleted by a pulse centrifugation in a microfuge, andthe supernatant was removed. The pellet was washed once a 70 μl of 100%ethanol, pelleted and resuspended in 30 μl of 100% ethanol. 8 μl of thetungsten/DNA suspension was spread and dried on the center of 1 inchdisc made of 2 mil plastic membrane (for ‘flying disc’ method), or a 1inch disc of 94 μm nylon mesh (for ‘helium entrainment’ method).

[0097] For the ‘flying disc’ configuration, the flying disc was thenloaded into a brass launch ring, which was screwed into a sleeve, with ametal screen on a retainer ring 1.3 cm below the brass launch ring. Theflying disc had an effective flight distance of 1 cm. For the ‘heliumentrainment’ configuration, the nylon mesh was trapped between two brassrings, which were screwed into the sleeve. The sleeve was then placedinto the sleeve holder at the desired height. The rupture-end of thehigh pressure chamber of the gun was sealed with an appropriate numberof isopropanol soaked 2 mil plastic rupture discs (3 layers for 900 psi;4 for 1200 psi and 5 for 1500 psi). The target sample was loaded at achosen platform height in the sample chamber, and bombarded underpartial vacuum (0.3 atmosphere).

[0098] In all experiments, at least 3 replicate petri plates werebombarded per treatment. After bombardment, 0.5 ml fresh NT1 medium wasadded to each plate. The plates were sealed and incubated at 26° C. inthe light. Two days after bombardment, an additional 0.4 ml of NT1medium was added to each plate.

Polyethylene Glycol (PEG)-Mediated Transformation of NT1 Protoplasts

[0099] NT1 protoplasts were prepared as described by Ye and Earle(submitted), except that 2% Cellulase-RS (Onozuka) was used in place ofCELF Cellulase in the enzyme solution. PEG transformation of protoplastswas performed according to Negrutiu et al., Plant Molec. Biol (1987)8:363-373. Protoplasts were cultured in NT1 protoplast culture mediumPaszty and Lurquin, Biotechniques (1987) 5:716-718 solidified with 1%Sea Plaque Low Melting Temperature agarose (FMC) in 60 mm petri dishesat room temperature in the dark.

[0100] β-Glucuronidase Assays Three days after bombardment, cells wereassayed for GUS activity by adding an aliquot of 1.5 ml of the substratemixture as described by McCabe et al., Bio/Technoloqy (1988) 6:923-926.The petri dishes were then sealed and incubated at 37° C. overnight. Thecells on the filter paper were observed with a microscope and the-numberof individual cells or aggregates of cells that developed blue colorwere counted. Transformation efficiency was expressed as number of bluespots per plate.

[0101] Chloroplast Expression of GUS

[0102] Cultured NT1 cells bombarded with either nuclear or chloroplastexpression vectors were assayed for the expression of GUS upon theaddition of a substrate mixture, three days after bombardment. Cellsbombarded with either pUC118 or pBI101.3 (negative controls) showed noGUS activity. Cells bombarded with pBI505 (positive nuclear control)showed high levels of expression (the highest being roughly 15,000transformants/plate) after overnight staining, and the blue color wasdistributed evenly throughout the whole cytosol of the stained cells. Incontrast, cells bombarded with pHD203-GUS (chloroplast promoter) did notshow GUS activity (blue spots) until after 4-5 days of staining. Enzymeactivity (blue color) was primarily localized subcellularly. Chloroplasttransient expression rates appeared to be 40-50 fold lower than nucleartransformation rates (the highest being 300-400 blue spots per petriplate). The expression of pHD203-GUS was maximal three days afterbombardment and dropped rapidly (FIG. 8), which is consistent with ourexperience with CAT expression of repliconless chloroplast expressionvectors (see above).

[0103] Confirmation of Organelle-Specific Expression of GUS

[0104] To confirm that pHD203-GUS was truly expressing in thechloroplast, we needed to verify that it did not express in the nucleus.Therefore, pUC118, pBI505 and pHD203-GUS were introduced into tobaccoNT1 protoplasts by PEG mediated transformation, followed by a GUS assay(FIG. 9). While pBI505 showed a high level of expression of GUS, no GUSactivity was observed for either the negative control, pUC118, or thechloroplast expression vector, pHD203-GUS.

[0105] Optimization of Chloroplast Transformtion

[0106] Osmoticum was found to have a major effect on transformationefficiency for both the chloroplast and the nucleus. When there was nosupplemental osmoticum in the cell medium, chloroplast transformationwas relatively low. As the concentration of osmoticum increased,transformation efficiency increased dramatically (about 20 fold) untilit reached a maximum near 1.0 M sorbitol/mannitol (FIG. 10). Furtherexperiments confirmed that 1.1 M sorbitol/mannitol was optimal fortransient expression of GUS in chloroplasts of cultured NT1 cells. 1.5 Mosmoticum significantly reduced transformation-efficiency. Theimportance of osmotic support of bombarded cells has also been seen withyeast Armalco, et al., Curr Genet (1990) 17: 97-103. However, theoptimal concentration of osmoticum for biolistic treatment variesgreatly, ranging from 0.25 M-1.75 M, depending on the species.

[0107] The helium biolistic transformation device increased chloroplasttransformation efficiency dramatically. The commonly used gun-powder anda new helium device were contrasted in parallel experiments. Chloroplasttransformation efficiencies were less than one transformant per petriplate for the traditional gun powder charge driven device, with the bestplate having eight transformants; in contrast, transformationefficiencies with the helium driven biolistic device were greater than200 transformants per petri plate, with the best plates having 300-400transformants.

[0108] Experiments were conducted comparing different launchconfigurations (flying disc vs. helium entrainment) at differentpressures, sample levels and sleeve levels (Table 1 below). These testswere made without supplemental osmotic support in the media, so: rateswere not optimal. In general, the helium entrainment configurationyielded more transient transformants than the flying disc configurationwith the same combinations of pressure, sample and sleeve levels. Forthe flying disc configuration, 900 psi pressure, 6.1 cm from targetcells to particle launch point, and the highest sleeve level (0.55 cmfrom rupture disc to flying disc) yielded the best results (an averageof 52 blue spots per plate). For the helium entrainment configuration,1500 psi pressure, 8.1 cm from target to launch point and the middlesleeve level (1.0 cm from rupture disc to nylon mesh) were optimal (anaverage of 227 blue spots per plate). TABLE 1 Effect of Configuration,Helium Pressure, Sample level And Sleeve Level in NT1 ChloroplastTransformation Efficiency. Sample Sleeve Configuration Pressure (Ph)Level¹ (cm) Level² (cm) Efficiency³ Helium 900 6.1 1.6 174 Entrainment6.1 0.55 134 8.1 1.0 124 1200 8.1 1.6 163 4.1 1.6 60 8.1 0.55 36 6.1 1.09 1500 6.1 1.6 59 6.1 0.55 46 4.1 1.0 2 8.1 1.0 227 Flying Disc 900 6.11.6 18 6.1 0.55 52 4.1 1.0 27 8.1 1.0 9 1200 8.1 1.6 2 4.1 1.6 5 4.10.55 4 8.1 0.55 4 6.1 1.0 1 1500 6.1 1.6 2 6.1 0.56 3 4.1 1.0 3 8.1 1.02

EXAMPLE IV Construction of Glyphosate Selection Vectors for StableChloroplast Transformation

[0109] The non-selective, broad spectrum herbicide glyphosate,N-phosphonomethyl)-glycine, interferes with aromatic amino acidbiosynthesis by inhibiting the shikimic acid pathway enzyme5-enolpyruvyshikimic acid 3-phosphate (EPSP) synthase Steinrucker andAmrehin Biochem. Biophys. Res. Commun. (1980) 94:1207-1212. Plant cellsuspension cultures can adapt to increases in the glyphosateconcentration in the growth medium by overproducing EPSP synthase. Geneamplification has been shown to be the basis of glyphosate tolerance incultured Petunia hybrida cells Shah et al. Science (1986) 233:478-481.However, a mutant-aroA gene from bacteria, that encodes EPSP synthasethat is less sensitive to glyphosate has also been reported inSalmonella typhimurium. Comai et al. Science (1983) 221:370-371.

[0110] A mutant EPSPS gene conferring a high level of resistance toglyphosate (PMON 894) from petunia is flanked at the 5′ end by 35Spromoter-enhancer elements and by T-DNA right border sequences at the 3′end. The EPSPS coding sequence-(including the transit peptide sequence)was excised from pMON 894 as a BqlII-SmaI fragment and was inserted intothe MCS of pHD203 at BamHI-SmaI sites. Transit peptide (TP) sequencescould not be deleted because of lack of proper restriction sites;however, this would not intefere with the function of EPSPS insidechloroplasts because TP would be naturally cleaved inside the plastids.The presence of TP did not interfere with EPSPS function in E. coli andwas also found to be useful in distinguishing petunia EPSPS fromendogenous EPSPS in E. coli.

[0111] In order to facilitate stable integration of EPSPS into thechloroplast genome, the 3′ end of the psbA gene (rest of the codingsequence) will be inserted into the MCS present at the 3′ end of EPSPcoding sequence. This will facilitate integration of EPSPS directly intothe psbA gene of the chloroplast genome. The distinct advantage of thisborder sequence would be that the EPSPS gene could be targeted into aknown gene whose product is essential only for auxotrophic growth (psbAgene product, 32 kDa herbicide binding protein, functions in thephotosynthetic electron transport chain). The disadvantage is that itwill not be possible to regenerate green photosynthetic transgenicplants using this vector. However, this problem can be overcome byinserting an additional psbA coding sequence 5′ to the left bordersequence, as described later for atrazine selection. This would ensurethat a new complete psbA gene is integrated into the ct genome, therebyenabling recovery of photosynthetic transgenic plants.

[0112] In order to increase the copy number of the introduced plasmid, ashort pea chloroplast DNA fragment containing a replication origin (seeabove) identified as a displacement loop (D-loop), that has been testedby in vitro and in vivo DNA replication studies, will be inserted intoappropriate restriction sites. This fragment will be inserted outsidepsbA border sequences in the chloroplast EPSPS vector so that it is usedsolely to amplify the copy number of the introduced plasmid and that itdoes not get integrated into the ct genome. Since this fragment is fromthe inverted repeat (IR) region (see above) it is unlikely that it wouldfacilitate integration due to a copy correction mechanism operatingbetween the inverted repeats (Blowers et al. The Plant Cell (1989)1:123-132.

Example V Construction of Atrazine Selection Vectors

[0113] The chloroplast gene psbA codes for the photosyntheticquinone-binding membrane protein Q_(B), which is the target of theherbicide atrazine. The mutant psbA gene from an atrazine resistantbiotype of Amaranthus hybridus has recently been modified by fusing itscoding region to transcription regulation and transit-peptide-encodingsequences for SSU of RuBis CO (Cheung et al. Proc. Nat'l Acad. Sci (USA)(1988) 85:391-395. The mutant psbA gene will be excised from pSSU-S-ATR(obtained from Prof. Bogorad) as a BamHI fragment that contains theentire mutant Q_(B) protein encoding region and sequences coding forsixteen amino acids beyond the most probable initiation codon ATG of theQ_(B) gene. This fragment will be made blunt (by filling in of therecessed 3′ termini using Klenow fragment of E. coli DNA polymerase I)and inserted into the unique SmaI site present in the MCS, at the 3′ endof the EPSPS coding sequence of pHD203-MK-EPSPS-TP (FIG. 1b). Plantstransformed with this vector, though not selected on double herbicides,would still have both resistant genes stably integrated; a foreign geneunselected for, flanked by ctDNA sequences has been stably integratedand maintained in the chloroplast chromosome of Chlamydomonas. Cheung etal., supra.

[0114] From the above it can be seen that expression of exogenous DNAsuch as foreign genes or mutant mature genes inserted into thechloroplast genome may be obtained. The above results demonstrate thatchloroplasts can be transformed efficiently and the exogenous genesexpressed resulting in an altered chloroplast phenotype. As evidenced bythe above disclosure, plant species having a modified genotype andphenotype and provided in which the chloroplast has been altereddirectly via its genome rather than indirectly by the use of modifiedproteins expressed via the nuclear genome and translocated into thechloroplast.

[0115] All publications and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0116] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A DNA construct comprising: as components, atranscriptional initiation region from a gene capable of expression in achloroplast joined to a heterologous DNA sequence encoding a polypeptideof interest, wherein transcription of said DNA sequence is regulated bysaid initiation region, and a transcriptional termination region.
 2. TheDNA construct according to claim 1, wherein said gene is a chloroplastgene.
 3. The DNA construct according to claim 1, wherein said gene is apsbA gene, rbcL gene or atpB gene.
 4. A chloroplast expression vectorcomprising: a transcriptional initiation region from a gene capable ofexpression in a chloroplast, a DNA sequence comprising at least onecloning site and a transcriptional termination region.
 5. The expressionvector according to claim 4, wherein said gene is a chloroplast gene. 6.The expression vector according to claim 4, wherein said gene is a psbAgene, rbcL gene or B gene.
 7. The expression vector according to claim4, wherein said cloning site is a multiple cloning site.
 8. Theexpression vector according to claim 4, wherein said transcriptionaltermination region comprises at least one of a ribosomal RNA T1 or aribosomal RNA T2 terminator.
 9. The expression vector according to claim4, further comprising: a heterologous DNA sequence encoding apolypeptide of interest inserted into said cloning site in reading framewith said transcriptional initiation region.
 10. A replication vectorcomprising: a DNA fragment comprising a replication origin capable ofproviding for autonomous replication in a chloroplast, a transcriptionalinitiation region from a gene capable of expression in a chloroplast, aDNA sequence comprising at least one cloning site and a transcriptionaltermination region.
 11. A chloroplast comprising: a DNA constructcomprising, as components, a transcriptional initiation region from agene capable of expression in a chloroplast joined to a heterologous DNAsequence encoding a polypeptide of interest, wherein transcription ofsaid DNA sequence is regulated by said initiation region, and atranscriptional termination region, wherein said components are operablylinked in vitro.
 12. A chloroplast comprising: a chloroplast expressionvector comprising, as components, a DNA fragment comprising areplication origin capable of providing for autonomous replication in achloroplast, a transcriptional initiation region from a gene capable ofexpression in a chloroplast, a DNA sequence encoding a polypeptide ofinterest and a transcriptional termination region, wherein saidcomponents are operably linked in vitro.
 13. A plant cell comprising: achloroplast according to claim 11 or claim
 12. 14. The plant cellaccording to claim 13, wherein said cell is monocotyledenous ordicotyledenous.
 15. A dicotyledenous plant comprising: cells containingchloroplasts according to claim 11 or claim
 12. 16. A method forintroducing heterologous DNA into a chloroplast, said method comprising:transforming a chloroplast in a plant cell with an expression vectorcomprising, as components, a transcriptional initiation region from agene capable of expression in a chloroplast, a heterologous DNA sequenceencoding a polypeptide of interest and a transcriptional terminationregion, wherein said components are operably linked in vitro.
 17. Themethod according to claim 16, wherein said expression vector furthercomprises: a DNA fragment comprising a chloroplast replication origin.18. A method for specifically modifying the phenotype of a chloroplastdistinct from other organelles, said method comprising: introducing intoa chloroplast in a plant cell, a chloroplast expression vectorcomprising, as components, a DNA fragment comprising a chloroplastreplication origin, a transcriptional initiation region from achloroplast gene, a DNA sequence encoding a polypeptide of interest anda transcriptional termination region, wherein said components areoperably linked in vitro and are functional in said chloroplast; andgrowing said cell whereby the phenotype is modified as a result ofexpression of said DNA sequence.
 19. The method according to claim 16,wherein said introducing comprises: bombarding said plant cell with aDNA construct comprising said expression vector adsorbed to abombardment particle.
 20. A chloroplast containing heterologous DNA,prepared according to the method of claim 19.